Patent Application
20100252750
The invention generally relates to imaging systems, and relates in particular to microscopy systems and methods.
Fluorescence microscopy has been widely used in biomedical sciences because of its high sensitivity and specificity. M any light-absorbing chromophores however, such as hemoglobin and cytochromes, have extremely low fluorescent quantum yields due to the much faster non-radiative decay rate than the spontaneous emission rate. In such cases, the remaining feeble fluorescence signal is overwhelmed by various background signals including stray light, solvent Raman background and detector dark counts, etc. Molecular contrasts other than fluorescence, therefore, would be highly beneficial for sensitive detection and imaging of these chromophores with non-detectable fluorescence.
Various types of fluorescence-free spectroscopy have been employed to image those chromophores, including photothermal (see “Label-Free Optical Imaging of Mitochondria in Live Cells” by D. Lasne, G. A. Blab, F. De Giorgi, R. Ichas, B. Lounis, and L. Cognet, Optics. Express vol. 15, no. 21, pp. 14184-14193 (Oct. 17, 2007)) and two-photon absorption (see “High-Resolution in vivo Imaging of Blood Vessels without Labeling” by Fu, D., Ye, T., Matthews, T. E., Chen, B. J., Yurtserver, G. & Warren, W. S., Optics Letters, vol. 32, no. 18, pp. 2641-2643 (Sep. 15, 2007)). These methods however, are still very limited in detection sensitivity.
The detection of single molecule absorption was previously achieved in cryogenic temperatures using frequency modulation (see “Optical Detection and Spectroscopy of Single Molecules in a Solid” By Moerner, W. E. & Kador, L., Phys. Rev. Lett. vol. 62, no. 21, p. 2535-2538 (May 22, 1989)). It is difficult however, to implement at room temperatures because of the broad absorption spectrum.
Surface enhanced Raman scattering (SERS) at electronic resonance has been achieved with single molecule sensitivity for those molecules having correct orientations with respect to metallic structures (see “Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering” by Nie S, Emory S R., Science, vol. 275, pp. 1102-1106, (Feb. 21, 1997)); and “Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)” by Kneipp K, Wang Y, Kneipp H, Perelman L T, Itzkan I, et al., Phys. Rev. Lett., vol. 78, no. 9, pp. 1667-1670 (Mar. 3, 1997)). The introduction however, of metal particles perturbs the sample and not all molecules in the sample can be accessed by SERS.
There is a strong need therefore, for a microscopy system and method for providing improved sensitivity in imaging chromophores, and in particular, for providing a microscopy system that permits imaging of light absorbing subjects having extremely low fluorescence.
The invention provides a microscopy imaging system in accordance with an embodiment of the invention that includes a light source system, focusing optics, an optical detector and a processor. The light source system is for providing an excitation beam at a center optical frequency ωe and for providing a stimulation beam at a center optical frequency ωs. The focusing optics is for directing and focusing the excitation beam toward a common focal volume such that an energy level of a sample may be excited to an electronic excited state, and for directing and focusing the stimulation beam toward the common focal volume such that stimulated emission induced from the electronic excited state results in an increase in intensity of the stimulation beam. The optical detector is for detecting an increase in a radiation field at the center optical frequency ωs from stimulated emission from the common focal volume and for providing a detector signal. The processor is for receiving the detector signal and for providing a pixel of an image for the microscopy imaging system.
The invention also provides a method of performing microscopy imaging that includes the steps of an providing excitation beam at a center optical frequency ωe, providing a stimulation beam at a center optical frequency ωs; directing and focusing the excitation beam toward a common focal volume such that an energy level of a sample may be excited to an electronic excited state; directing and focusing the stimulation beam from the stimulation illumination toward the common focal volume such that stimulated emission induced from the electronic excited state results in an increase in intensity of the stimulation beam; detecting an increase in a radiation field at the center optical frequency ωs from stimulated emission from the common focal volume; providing a stimulated emission detector signal responsive to the increase in the radiation field at the center optical frequency ωs from stimulated emission from the common focal volume; and providing at least a portion of an image responsive to the stimulated emission detector signal.
In certain embodiments, the stimulated emission imaging of the invention allows detection and imaging of non-fluorescent chromophores such as drug molecules, small dye molecules and proteins in living cells, tissues and organisms with intrinsic 3D optical sectioning and high sensitivity.
The following description may be further understood with reference to the accompanying drawings in which:
The drawings are shown for illustrative purposes only.
Fluorescence is a powerful contrast mechanism used in molecular imaging due to its high sensitivity. Many light-absorbing chromophore molecules however, are only weakly fluorescent, because of their fast non-radiative decay. The feeble fluorescence from such chromophores is often overwhelmed by various background signals including stray light, solvent Raman background and detector dark counts, etc. Various fluorescence-free techniques have been developed, but are often limited by their weak signals.
The present invention provides a new contrast mechanism for room temperature imaging systems that is based on stimulated emission. The radiative emission rate from the molecular excited state is significantly amplified by virtue of stimulated emission, which converts the originally non-, or weakly radiating species into highly radiating. The superb sensitivity is accomplished by implementation of high-frequency (MHz) phase-sensitive detection. The overall nonlinear intensity dependence of the stimulated emission signal also offers an intrinsic three-dimensional optical sectioning capability.
For example, in accordance with certain embodiments, the invention provides orders-of-magnitude improvement of detection sensitivity for non-fluorescent chromophores by use of stimulated emission that dominates the non-radiative decay. In a femtosecond pump-probe experiment, shortly after optical excitation by the pump pulses, the probe pulses stimulate the transition from the molecular excited state down to the ground state, and at the same time, experience a light amplification after passing through the molecules. Such a stimulated emission signal is extracted by implementing high-frequency (MHz) phase sensitive detection with high sensitivity. The resulting signal is linearly dependent on both the pump and probe intensities, offering intrinsic three-dimensional optical sectioning capability for microscopy. A variety of applications of this technique are demonstrated, such as visualizing distributions of chromoproteins, non-fluorescent variants of the green fluorescent protein, in live bacteria, monitoring basal level lacZ gene expression based on chromogenic substrate, 3D optical sectioning of medically stained tissues, and imaging subcellular distribution and transdermal delivery of a drug used in photodynamic therapy. The microscopic technique also opens up the possibility for studying the biochemistry of endogenous proteins such as cytochromes and hemoglobin without labeling.
The phenomenon of stimulated emission was first described by Albert Einstein in 1917 in term of Einstein's B coefficients. An atom or molecule in its excited state can be stimulated down to the ground state by an incoming light field, resulting in the creation of a new photon identical to those in the incoming field. This process only occurs when the frequency of the incoming field matches the energy gap between the ground and the excited state. Stimulated emission is the basis for light amplification in laser. The depopulation aspect of stimulated emission has been successfully used for population dumping from molecular excited states, super-resolution fluorescence microscopy, and fluorescence lifetime imaging. The present invention utilizes the light amplification aspect of stimulated emission as a contrast mechanism for high-sensitivity microscopy.
The minimal spontaneous emission from weakly fluorescent chromophores, is overwhelmed by various background signals, such as stray light, solvent Raman scattering, detector dark counts, etc. due to the non-radiative decay rate being much faster than the spontaneous emission rate (i.e., Einstein's A coefficient). Applicants have discovered that a solution to this problem is to probe the short lived excited state by stimulated emission that dominates the non-radiative decay. In a pump-probe experiment, shortly after photo-excitation of the chromophore, stimulated emission is induced by a stimulation pulse during the short excited state lifetime, resulting in an increase in the stimulation beam's photon flux, which can be detected against the background. The approach of the present invention introduces an external coherent laser field to greatly stimulate the radiative emission from the electronic excited state after the chromophore is optically excited but before its non-radiative decay dominates.
The invention, therefore, involves stimulating emission of non-fluorescent or weakly fluorescent samples at an electronic excited state. As shown in the energy diagram in
The invention provides that prior to the non-radiative decay in a non-fluorescent or weakly fluorescent sample, a stimulated emission may be extracted as shown at 24 from the energy state 16, which is the relaxed state of the higher energy level, to the energy state 20. In accordance with an embodiment, consecutive optical excitation at one wavelength ω01 and stimulated emission at a longer wavelength ω23 may be provided. Spontaneous emission is much slower than the non-radiative decay in weakly or non-fluorescent chromophores. When the stimulation field is designed to have the correct energy and timing, the stimulated emission can be the dominating decay pathway.
The excitation field and stimulation field may be provided as a stimulation beam 30 and an excitation beam 32 as shown in
With reference again to
The molecular absorption cross section σabs for a single chromophore in solution at room temperature is ˜10−16 cm2. Under a tightly focused laser beam with a beam waist area of S (˜10−9 cm2 for visible light focused by a high numerical aperture objective), the integrated intensity attenuation of the excitation beam, ΔIE/IE, is proportional to the ratio between σ0→1 and S:
ΔIE/IE≈−N0·σ0→1/S (1)
where N0 is the number of molecules in ground state. For a single chromophore, i.e. N0=1, ΔIE/IE is on the order of 10−7. Attenuation magnitude at such a scale cannot be detected by conventional absorption microscopy. It is noted that single molecule absorption has been previously achieved in cryogenic temperatures using frequency modulation, which is difficult to implement because of the broad absorption spectrum at room temperatures. Instead of detecting direct absorption, the invention provides detecting stimulated emission followed by absorption.
The molecular cross section σsti.em for stimulated emission, which is proportional to Einstein's B coefficient, is comparable to σabs. Similarly, the intensity gain of the stimulated emission beam, ΔIS/IS, is as follows
ΔIS/IS≈N2·σ2→3/S (2)
where N2 is the number of excited molecules interrogated by the stimulation pulses. For a single chromophore residing in level 2, i.e., N2=1, ΔIS/IE is also on the order of 10−7.
Such a small amplification is again often buried in the laser noise (˜1%) of the stimulated emission beam. By implementation of a high-frequency (higher than MHz) intensity modulation technique however, the laser noise, which occurs primarily at low frequency (kHz to DC), may be sufficiently suppressed.
In the dual beam scheme, N2 in Equation (2) above originates from linear optical excitation: N2 ∝ N0·IE·σ0→1/S. This relation, together with Equation (2), indicates that the final signal ΔIS is linearly dependent on both IE and IS, i.e.,
ΔIS∝N0·IE·IS·(σ0→1/S)·(σ2→3/S).
The detected stimulated emission signal depends on the product of the excitation beam intensity and the stimulated beam intensity. The signal, therefore, has an overall second order nonlinear intensity dependence, which provides high spatial resolution.
With reference to
In specific examples, 200 fs pulses may be used for excitation and stimulation as they are shorter than the excited state lifetime (sub-ps) of certain chromophores. The stimulation pulses may be delayed with respect to the excitation pulses by ˜200 fs in order for the vibrational relaxation to complete from level 1 to level 2 (shown at 14 and 16 in
In particular, the intensity of the excitation beam is modulated, e.g., at 5 MHz, and this creates a modulation of the stimulated emission signal at the same frequency, because only when the excitation beam is present can the gain of the stimulated beam occur. Such an induced modulation signal can be sensitively extracted by the lock-in amplifier at 5 MHz, at which the laser noise is lower than 10−7. In this way, the dual beam modulation transfer scheme herein offers a superior sensitivity over the direct one-beam absorption detection.
The temporal delay between excitation and stimulation pulses is adjustable in certain embodiments by using a delay unit such as a translational stage for either one of the excitation and stimulation trains of pulses. In other embodiments, the delay may be provided within the laser source system itself that produces the excitation and stimulation trains of pulses.
Two femptosecond (fs) optical parametric oscillators (OPO), for example, may be synchronously pumped by a fs mode-locked 76 MHz Ti:Sapphire laser. Two frequency-doubled outputs from two OPO signal waves (in the near infrared range), in the wavelength range of 560 to 700 nm and pulse width around 200 fs, may provide the excitation and stimulation pulse trains, respectively. The excitation train of pulses is modulated by a modulator 68, and a modulated excitation train of pulses 70 is combined with the stimulation train of pulses 66 at a combiner 72.
The timing of the stimulation train of laser pulses 66 may be adjusted with respect to the timing of the modulated excitation train of laser pulses 70 by a delay unit 74 that is adjustable as shown at 76. The modulator 68 may, for example, be an acousto-optic modulator that switches the excitation train of pulses on and off at 5 MHz. The combined modulated excitation train of pulses and stimulation train of pulses 78 are provided to a microscope 80.
The microscope 80 includes optics 82 and a reflector system 84 for directing the combined pulses 78 toward an objective 86. The collinear modulated excitation and stimulation beams are focused with a high numerical aperture (N.A.) objective (NA=1.2) onto the common focal spot. T he temporal delay between the synchronized excitation and stimulation inter-pulse is adjusted to about 0.2 ps by using a translational stage. The intensity of the excitation beam is modulated by an acoustics optical modulator at 5 MHz. A condenser with a N.A.=0.9 is used to collect the forward propagating stimulation beam. To acquire images with laser beam scanning, we used a 100 μs time constant for lock-in amplifier and pixel dwell time of 190 μs.
In certain embodiments, the reflector system 84 may include x and y direction scanners (such as mirrors or a scanning light modulator) for scanning in x and y directions on a sample 88. In other embodiments, a stage on which the sample 88 is placed may be adjustable in x and y directions. In certain embodiments, the objective 86 may permit scanning in the z direction.
The tightly focused combined modulated excitation train of pulses and stimulation train of pulses is directed toward the sample 88, and illumination from the sample 88 is collected by lens 90 and filtered by filter 92 (which removes illumination at the excitation frequency), providing filtered illumination 94 that is received by a detector 96 such as a large-area photodiode.
A lock-in amplifier 98 is coupled to both the modulator 68 and the detector 96 such that the modulation may be employed by the detector 96 to identify via image contrast the illumination of interest from filtered illumination 94. The detector 96 provides a detector signal to a processing unit 100, which provides pixel data for an imaging system.
While the filter 92 and detector 96 are located in the forward direction with respect to the objective 86, in further embodiments, the detector and filter may optionally be located in the reverse (epi) direction with respect to the objective 86. For example, as also shown in
Each excitation pulse from the modulated train of excitation pulses causes chromophores in the sample to change energy states from the low (or ground) state to the electronic excited state, and a quickly following stimulation pulse from the train of stimulation pulses stimulates emission, causing the energy to be released as illumination at the excitation frequency, increasing the total radiative quantum yield by as much as from 10−5 to unity. As a result, the originally weakly or non-fluorescent species are turned into highly radiating species.
For example,
The measured temporal and spectral dependence of the stimulated emission signal were therefore experimentally confirmed. The time-delay dependence was found to be asymmetric as shown in
As shown at 140 in
This superb sensitivity in the nano-Molar range (approaching the shot noise limit) corresponds to about a few (<5) molecules within the focal volume of the microscope objective (˜10−16 liter). To detect higher concentration samples, laser power levels may be lowered to reduce photo-bleaching.
Imaging of live cells has been achieved using stimulated emission systems and methods of the invention.
The genetically encodable chromoprotein, such as gtCP and cjBlue, are variants of green fluorescent proteins, and only absorb light but do not fluoresce. When the gene encoding for gtCP is expressed in live E. coli cells, tetrameric gtCP may be clearly shown to reside evenly inside cytoplasm by stimulated emission microscopy, which clearly distinguishes bright colored (e.g., amber colored) areas 152 from the background 154 as shown in
Similarly, when the gene encoding for cjBlue is expressed in live E. coli cells, the cjBlue may be clearly shown to reside evenly inside cytoplasm by stimulated emission microscopy, which clearly distinguishes bright colored (e.g., blue colored) areas 162 from the background 164 as shown in
Since its discovery, lacZ has been a classic reporter for gene expression in various prokaryotic and eukaryotic cells. The protein product, β-galactosidase, encoded by lacZ gene, catalyzes the hydrolysis of X-gal, a popular chromogenic substrate, to form a bluish product. Traditionally, the X-gal hydrolysis product has to accumulate enough for its blue color to be visually seen. With stimulated emission, the basal level lacZ gene expression in the absence of inducer can now be sensitively monitored. Different from the homogeneous chromoprotein images, the more inhomogeneous distribution of X-gal hydrolysis product inside cells (shown in
Applicants have also discovered that the overall quadratic power dependence as outlined above (and as experimentally demonstrated), would allow three-dimensional (3D) optical sectioning, as in many other multi-photon techniques.
Imaging medically stained tissues with intrinsic 3D optical sectioning is, therefore, another suitable application for systems of the invention. Various types of chromophore staining are widely used in histology for medical diagnosis. For example, hematoxylin is wisely used to stains basophilic structures such as nuclei. In the conventional approach, thin (˜micron scales) sections have to be physically cut piece-by-piece, because the traditional wide-field transmission microscopy relies on linear absorption and thus does not have optical sectioning ability. Thanks to the nonlinear intensity dependence, stimulated emission microscopy can selectively show images at different depths of stained tissues because the signal is only generated at the laser focus where the laser intensity is the strongest.
Drug delivery of toluidine blue O (TBO), a drug used as photosensitizer in photodynamic therapy, is shown in
The use of stimulated emission microscopy to monitor drug delivery is therefore demonstrated. In particular, we show mapping of a cationic thiazine dye toluidine blue O (TBO) at both the cellular and tissue levels. Having a selective affinity for cancer cells in vivo, TBO is an actively explored photosensitizer in photodynamic therapy. Subcellular localization of photosensitizers is crucial since it can influence both the level and the kinetics of apoptosis induction. It is conventionally difficult, however, to image the true distribution of TBO because its fluorescence is quenched when bound to tissue substrates and only the non-specific stain residue in the tissue retains its native fluorescence. Because stimulated emission microscopy is independent of fluorescence contrast, it is suitable for addressing this problem.
The stimulated emission image of TBO inside cancer cells after incubation clearly shows its local accumulation inside cytoplasm instead of membrane or nucleus. When topically applied to skin tissue, being hydrophilic and water soluble, TBO is enriched in the center of the protein phase of the polygonal stratum corneum cells rather than in the intercellular space which is in lipid phase. At a 20 μm deeper depth, TBO shows a rich distribution following the subcellular cytoplasm of nucleated viable epidermis in which cellular proliferation actively takes place. These imaging results are consistent with the known high affinity of TBO for cytoplasmic RNA.
Stimulated emission microscopy, therefore, allows detection and imaging of non-fluorescent chromophores such as drug molecules, small dye molecules and proteins in living cells, tissues and organisms with intrinsic 3D optical sectioning and high sensitivity.
Those skilled in the art will appreciate that numerous modifications and variations may be made to the above disclosed embodiments without departing from the spirit and scope of the invention.
